R2 value was established as 0.987 and k2 was .02671. The greater R2 value of second-order kinetics over first-order kinetics suggested that MR adsorption by nanocomposite is best explained by pseudo-second-order kinetics, indicating that dye adsorption occurred through chemisorption. The R2 value was determined to be .9954. The correlation coefficient values of Gk-cl-poly(AA)-AgNPs were best fitted by the Freundlich adsorption isotherm. Overall, synthesized bionanocomposite is a proficient material for removing of MR dye from wastewater.The present study aimed to synthesize gum katira-silver nanoparticle-based bionanocomposite. Different characterization techniques were used to analyze the synthesized bionanocomposite, such as Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), thermo-gravimetric analysis (TGA), and transmission electronic microscopy (TEM). AgNPs were formed and were 6–20 nm in size. Thermo-gravimetric analysis showed that synthesized nanocomposites are more thermally stable than gum katira. All the reaction conditions, such as time, temperature, pH, solvent, amount of nanoparticles, the concentration of the initiator, crosslinker, and monomer were optimized with respect to swelling. The results showed that the highest percentage swelling (Ps) of Gk-cl-poly(AA) was 796%, and 867% of AgNPs were imbibed by Gk-cl-poly(acrylic acid)-AgNPs. Synthesized bionanocomposite was used as an adsorbent material for the adsorption of methyl red (MR) dye. The effects of different reaction conditions were also optimized to attain maximum adsorption of MR dye. The maximum dye adsorption through Gk-cl-poly(AA)-AgNPs bionanocomposite was 95.7%. Diverse kinetic and isotherm models were used to study the adsorption data. The  Water is precious for all forms of life and different industries must treat it well before discharging it. Owing to advancements in lifestyle, industrial activities have increased at a great pace and the environment has gravely depreciated as a result, particularly the aquatic environment. Toxic organic pollutants from industrial effluents damage the environment, and dyes are the foremost pollutants that contaminate water bodies and are easily recognizable by eye . As thesThere are different types of processes for the removal of harmful dyes from industrial effluents, such as oxidation, electro-coagulation, solvent extraction, trickling filtration, ion exchange, activated sludge, and the use of microorganisms . These pNanocomposites are a class of material that includes a minimum of one constituent in the nanometric range. They are labeled as the materials of 21st century due to their unique design and properties, which are different from synthesized materials. They are inventive materials that possess different properties from their constituents . Many revia Ca+2 crosslinking and a freeze-thaw method. The synthesized material was used as an adsorbent for the removal of the harmful dye MB. Later, the synthesized hydrogel was functionalized with activated carbon, Fe3O4, and graphene oxide. The Fe3O4-containing composite is utilized as a heterogeneous Fenton catalyst to degrade MB. The isotherm was best fitted by the Langmuir adsorption isotherm  (backbone), Ammonium persulfate (APS) (initiator), N,N′– methylenebisacrylamide (MBA) (crosslinker), acrylic acid (AA) (monomer), and silver nitrate were procured from Loba Chemie Pvt Ltd., Mumbai, India. All chemicals were used as received. All solutions were prepared in double-distilled water.In a typical experiment, 1 g of gum katira was dissolved in 10 ml of deionized water at ambient temperature to homogenously dissolve the biopolymer. The required amount of acrylic acid was added and stirred for 1 h. Then, the required amounts of MBA and initiator APS were added. A specific amount of ammonium persulfate, as well as MBA, was added to the reaction mixture and continuously stirred until the mixture was uniform and homogeneous. The reaction mixture was then warmed to the required temperature, stirred for a specific amount of time, and poured into acetone, and the precipitated grafted guar gum was isolated by filtration. The mixture was then washed twice with acetone, filtered, and dried at 60°C in an oven until it reached a constant weight. Every single reaction parameter, such as time, temperature, pH, solvent, initiator, crosslinker, and monomer, was optimized with respect to the percentage of swelling (Ps). The homopolymer that formed was separated by extraction with deionized water, and then Gk-cl-poly(AA) was dried .Gk-cl-poy(AA) (1 g) was solubilized in 10 ml of water; .1 M NaOH solution was used to maintain a pH of 10. Silver nitrate (1 mmol) solution was added slowly with stirring and the mixture was then heated for 5 min. The color of the solution immediately changed to brownish-yellow, which confirmed the development of AgNPs. Gk-cl-poly(AA)-AgNP bionanocomposite was taken out, washed, and dried until a constant weight was obtained .s and Wd are the weight of the swollen and dried sample, respectively.The swelling of synthesized Gk-cl-poly(AA) and Gk-cl-poly(AA)AgNPs was studied. A specific amount of synthesized Gk-cl-poly(AA) and Gk-cl-poly(AA)AgNPs was submerged in 100 ml of deionized water. Then, after a certain period of time, Gk-cl-poly(AA) was removed, wiped softly, and weighed. The percentage of swelling (Ps) in swollen Gk-cl-poly(AA) was determined using the following equation :Ps=Ws−W−1). The morphological variations were studied using a high-resolution SEM . Samples were dried and gold plated for SEM characterization. The thermal behavior of the samples was studied using TGA/DTA 6300 and SII EXSTAR 6000 instruments . TGA scans were recorded from 50°C to 700°C at a heating rate of 10°C per minute. Nanoparticle sizes were investigated using a transmission electron microscopy (TEM) . TEM samples were prepared on a carbon film supported by a copper grid.FTIR spectra were recorded with a Bruker Alpha FTIR spectrometer  using KBr pellets   are acrylic acid and MBA. It is clear from the FTIR results that grafting and crosslinking occurred in the backbone and monomer.The FTIR of Gk and Gk-cl-poly(AA) was analyzed using a PerkinElmer spectrophotometer. poly(AA)  illustraThe exterior morphology of Gk, Gk-cl-poly(AA), and Gk-cl-poly(AA)-AgNPs was examined by SEM. A smooth surface was observed in gum katira . The surTGA evaluates the mass of Gk, Gk-cl-poly(AA), and Gk-cl-poly(AA)-AgNPs, while heating, cooling, or a stable temperature represents the stoichiometric decomposition of components and the mass of substance missing at different steps. Combustion products generated by burning of the samples escape into the atmosphere and the mass of the substance decreases leaving behind ash.Gk was heated up to 270°C. The thermal stability of SIPN was observed up to 105°C, at which point volatile substituents were lost. In the second step, further pyrolysis took place up to 235°C, at which point the decomposition process caused the release of carbon monoxide and carbon dioxide. Once temperatures rose above 235°C, a black residue was left behind. Polymer content weight loss during the different steps was as follows: 30.9% during the first step and 68.6% during the second step .As depicted in The decomposition of Gk-cl-poly(AA)-AgNPs occurred at 380°C. During the first step, sample burning was accompanied by the escape of some volatile components up to 80°C. During the second step, heating continued up to 220°C, further advancing the decomposition process. During the third step, the combustion of carbon took place up to a temperature of 330°C. At 330°C, the combustion of carbon black additives took place. Some amount of residue was left behind at 380°C. The loss of weight of the sample during thermal analysis was 31.97% during the first step, 27.58% during the second step, and 40.60% during the third step . These rParticle size and shape was analyzed by TEM, which allowed the distinctiveness of very small-sized samples to be observed. The synthesized AgNPs were nanosized and clearly spherical and ellipsoidal in shape .Ps increases as reaction time increases and a maximum swelling of 596%  was atta.The impact of temperature was investigated as it is an important parameter for grafting. The highest Ps was obtained at 60°C (426%) . When thThe highest Ps was obtained at a neutral pH (312%) . There aPs increased with an increase in APS concentration, and optimum Ps (796%)  was obta− to markedly increase to a level that terminates the polymerization reaction, thus decreasing Ps .Ps increased as the amount of solvent increased, and a maximum Ps of 736% was attained using 14 ml of solvent . The con−1 (The highest Ps (318%) was obtained at a monomer concentration of 4.1632 molL−1 . Ps decr−1 of MBA (The highest Ps (459%) was observed with .0139 molL1 of MBA . Ps incrThe amount of nanoparticles (AgNPs) added during the preparation of Gk-cl-poly(AA)-AgNP bionanocomposite varied from .01 to .05 g. The percentage of swelling increased from .01 to .03 g and then decreased at higher levels, exhibiting a maximum swelling of 867% at .03 g of nanoparticles .MR dye removal was slow at the early stage, but with time, dye removal increased. An equilibrium was attained at 20 h, at which point dye removal was at its maximum (92%) . PrimariThe initial concentration of the dye had a significant impact on their eradication; therefore, differing concentrations of dye solution (10–50 ppm) were examined with respect to the percentage of dye removal. The highest percentage of MR dye removal was 95.6% at 10 ppm ; higher pH is an important factor in the chemistry between the adsorbent surface and the dye molecule. Electrostatic interaction takes place between adsorbate and adsorbent. Dye extraction increased as pH increased, and equilibrium was attained at pH 10. No further increases in the adsorption of dye were observed beyond pH 10 due to occupancy of active sites. The highest percentage of dye removal (92%) occurred at pH 10 .The outcome of formulated Gk-cl-poly(AA)-AgNP dose on MR dye removal was examined at a range of doses (.1–0.6 g) . The adsAdsorption kinetics play an important role in the relationship between the time required for adsorption and the dye molecule exclusion rate. Kinetic studies were carried out at various time intervals using pseudo-first- and pseudo-second-order kinetics. Result showed that initially there is an increase in dye uptake but eventually an equilibrium is reached, after which dye uptake decreases. The abundance of adsorption sites accounts for the initial dye adsorption, but later these active sites become exhausted, and thus, a constancy is reached .−1), qt is the adsorption capacity at time t (mg g−1), and k1 refers to the pseudo-first-order rate constant (g mg−1min−1)  . Log qe-tal data .e and qt refer to the amount of dye adsorbed at equilibrium and at time t, whereas k2 represents the second-order rate constant  is the equilibrium concentration of MR dye, qe represents the quantity of adsorbed dye molecules at equilibrium (mg g−1), b is the Langmuir constant, and Qm is the monolayer capacity. The values of b and Qm were determined from the intercept and slope from the plot of 1/qe vs. Ce. The R2 value was .7184 , the regeneration efficiency was 88.6%, 79.3%, 68%, and 53% in the first, second, third, and fourth cycles, respectively. It was clear from the results that synthesized bionanocomposite was stable and retained its adsorption capacity for longer. Therefore, synthesized bionanocomposite is an effective and efficient adsorbent for water treatment .The stability of the synthesized AgNPs in the Gk-cl-poly(AA) was investigated by immersing .2 g of Gk-cl-poly(AA)AgNP bionanocomposite in 20 ml of distilled water, which was then vigorously shaken in a water-bath shaker  for different time periods. After a specific period of time, the sample was checked for the quantity of AgNPs leached into the water using a UV-Vis spectrophotometer at 432 nm. It was clear from the results that AgNPs leached into the water in very small amounts (.1 μg/L) after 1 h and increased to .35 and .42 μg/L after 4 and 12 h, respectively. The acceptable concentration for Ag in drinking water, according to the WHO, is 8–49 μg/L. Thus, it can be concluded from the above results that it is safe to use the bionanocomposite as an adsorbent.The efficiency of synthesized bionanocomposite was compared with the published literature. Green synthesizes of bimetallic Ag/ZnO@biochar nanocomposite showed a photodegradation efficiency of up to 70.3% . BiogeniR2 and k2 values were .98783 and .02671, respectively. The greater R2 value of second-order kinetics compared with first-order kinetics suggested that MR adsorption by nanocomposite is best explained by pseudo-second-order kinetics. The foregoing discussion has shown that Gk-cl-poly(AA)-AgNPs are an effective adsorbent for the removal of the harmful dye MR and is eco-friendly in nature. This adsorbent can be used in the future as a tool for removing dye pollution from water.The present study demonstrated the synthesis of a novel gum katira and a silver nanoparticle-based bionanocomposite. TEM images confirmed the formation of silver nanoparticles. FTIR and SEM confirmed that grafting occurred in the gum katira and acrylic acid. Grafting and crosslinking made the synthesized bionanocomposite thermally stable. The maximum percentage swelling of Gk-cl-poly(AA) and Gk-cl-poly(AA)-AgNPs was 796% and 867%, respectively. The highest dye adsorption from Gk-cl-poly(AA)-AgNPs was 95.6%. The


Optogenetic techniques were used to verify the neural mechanisms underlying the behavioral effects induced by DBS. Our results showed that electrical stimulation of the VTA activates local gamma-aminobutyric acid (GABA) neurons, and dopamine (DA) neurons, reduces hyperlocomotion, and relieves stereotyped behaviors induced by MK-801 (dizocilpine) injection. The results of optogenetic manipulation showed that the activation of the VTA GABA neurons, but not DA neurons, is involved in the alleviation of hyperlocomotion and stereotyped behaviors. We visualized changes in the activity of specific types in specific brain areas induced by DBS, and explored the neural mechanism of DBS in alleviating positive psychotic-like behaviors. This preclinical study not only proposes new technical means of exploring the mechanism of DBS, but also provides experimental justification for the clinical treatment of psychotic diseases by electrical stimulation of the VTA.Deep brain stimulation (DBS) is a clinical intervention for the treatment of movement disorders. It has also been applied to the treatment of psychiatric disorders such as depression, anorexia nervosa, obsessive-compulsive disorder, and schizophrenia. Psychiatric disorders including schizophrenia, bipolar disorder, and major depression can lead to psychosis, which can cause patients to lose touch with reality. The ventral tegmental area (VTA), located near the midline of the midbrain, is an important region involved in psychosis. However, the clinical application of electrical stimulation of the VTA to treat psychotic diseases has been limited, and related mechanisms have not been thoroughly studied. In the present study, hyperlocomotion and stereotyped behaviors of the mice were employed to mimic and evaluate the positive-psychotic-like behaviors. We attempted to treat positive psychotic-like behaviors by electrically stimulating the VTA in mice and exploring the neural mechanisms behind behavioral effects. Local field potential recording and  The clinical application of deep brain stimulation (DBS), a neurosurgical procedure commonly used to treat movement disorders such as Parkinson’s disease, tremor, and dystonia, has been one of the most important advances in clinical neuroscience in the last two decades  is an important brain region involved in the onset and etiology of psychosis  C57BL/6 male mice (Shanghai Model Organisms), Vgat-ires-cre knock-in mice (Stock No. 028862) and DAT-ires-cre knock-in mice . The engineered mice were both maintained on a C57BL/6J genetic background.The care and use of animals were conducted in strict accordance with institutional guidelines and governmental regulations. All mice were maintained under a reversed 12-h /12-h day/night cycle at 22–25°C with All experiments involving mice were carried out in accordance with the US National Institutes of Health Guide for the Care and Use of Animals under an Institutional Animal Care and Use Committee approved protocol and Association for Assessment and Accreditation of Laboratory Animal Care approved Facility at the ShanghaiTech University.in vivo LFP electrodes  were slowly implanted into the VTA unilaterally or bilaterally . Then the optical fiber, DBS electrodes, in vivo LFP electrodes, and skull were fixed with dental cement. After the dental cement was completely dried, the mice were removed from the positioning instrument and placed on an electric blanket. After the mice fully recovered, they were put back into the feeding cage.After the mice were deeply anesthetized with isoflurane, they were placed on a stereoscopic positioning instrument. The eyes were coated with aureomycin eye cream, and the scalp was cut open. Then, the skull surface was wiped with 3% hydrogen peroxide to remove the fascia from the skull surface. The Bregma point and Lambda point were used to adjust the mouse head to the horizontal position. A small window with a diameter of 300–500 microns was opened at the location of viral injection and fiber implantation. According to the brain atlas, the ML range of the VTA is ±0.2–1.2 mm. Since both electrical stimulation and illumination stimulation act on the neurons within a certain range, we chose ±0.5 and ±0.7 mm, which are closer to the center, as the experimental coordinates. The viral injection rate was 300 nl/min. AAV2/9-hsyn-DIO-GCaMP6(m) was injected unilaterally for fiber photometry . rAAV-EF1a-DIO-hChR2(H134R)-mCherry/rAAV-EF1a-DIO-eNpHR3.0-mCherry was injected bilaterally for optogenetic experiments . For bilateral viral injection in the VTA, the syringe was angled 8° laterally to avoid the central sinus. After the viral injection, the apparatus remained in place for at least 10 min. Then, the fiber , the DBS electrodes , and the DAT-ires-cre and Vgat-ires-cre mice through intra-VTA injection of AAV2/9-hsyn-DIO-GCaMP6m. Following AAV-DIO-GCaMP6m viral injection, an optical fiber  was placed in a ceramic ferrule and inserted toward the VTA through the craniotomy. Mice were individually housed for at least 2 weeks to recover. Fluorescence signals were acquired with a fiber photometry system equipped with a 488 nm excitation laser, 505–544 nm emission filter, and a photomultiplier tube . The analog voltage signals were digitized at 100 Hz and recorded by a Power 1401 digitizer and Spike2 software . An optical fiber  with an integrated rotary joint preventing fiber damage from the animal movement was used to guide the light between the fiber photometry system and the implanted optical fiber. The laser power was adjusted at the tip of the optical fiber to the low level of 20–40 μW, to minimize bleaching.GCaMp6m expressed in VTA DA and GABA neurons using 0)/F0, where F0 is the baseline fluorescence signal averaged over a 5 s-long control time window. ΔF/F values were presented with average plots to illustrate the signal changes trial by trial. To calculate the average response and decrease the duration of ΔF/F values, we first segmented the data based on the behavioral events and baseline phase. Then we calculated the average calcium signal both in baseline and event phases.For the fiber photometry experiments, the analysis methods were as follows. Photometry data were exported to MATLAB R2020b mat files from Spike2 for further analysis. We segmented the data based on behavioral events within individual trials. The event time was recorded manually at every point of the stimulation. We derived the values of fluorescence change (ΔF/F) by calculating -mCherry and rAAV-EF1a-DIO-eNpHR3.0-mCherry were injected into the VTA. Two optical fibers were implanted above the VTA. After 3 weeks of virus expression, the experiments were carried out. First, the mice were allowed to adapt to the open field for 10 min. For mice expressing eNpHR3.0, a 555-nm yellow light laser was delivered continuously for 180 s. Light intensity was calculated to be about 10 mW. The interval between the two stimulations was 10–15 min. For mice expressing ChR2, first, we injected 0.3 mg/kg MK-801 and then placed the animal in the open field environment. Twenty minutes later, we began to activate the neurons with a 473-nm blue light laser delivered at 20 Hz in 15 ms pulses for 180 s. Light intensity was calculated to be about 10 mW. The interval between the two stimulations was 10–15 min.DBS stimulation: electrical current .Electrical stimulator: STG4002, Germany.In our study, the current polarity is bipolar stimulation. There are two electrodes on each side of the VTA, which are connected to the positive and negative poles of the stimulator respectively. The metal electrode at 0.5 mm of the tip of the electrode is exposed and the rest is covered by an insulating layer. One electrode serves as the cathode while another serves as the anode.Mice were unilaterally implanted with a 75-μm stainless-steel electrode  in the VTA . They were anesthetized with isoflurane for the implantation procedure  and allowed to recover for 1 week after surgery. Recording signals (low-pass filter: 1–100 Hz) were digitized by the Ephyslab System (Thinker Tech Nanjing Biotech Co. Ltd.) at 30 kHz, and then resampled at 1 kHz for the LFP analysis.During the recording process, LFPs of the VTA neurons were recorded under normal conditions as a baseline value. MK-801 (0.3 mg/kg), which takes 30 min to exert its effects, was intraperitoneally injected into the mice as a pharmacological model of psychosis. The LFPs of the VTA in the last 5 min of the 30 min post-administration period were recorded as the pathogenic values under pathological conditions. Next, the VTA was stimulated with a 60 μs wave width, 130 Hz, and 100 μA current for 1 min. After the stimulation stopped, LFPs were recorded for 5 min as the therapeutic value. Finally, MATLAB R2020b was used for statistical analysis. Target oscillations  were classified based on previous studies.The videos were recorded simultaneously with a camera and the LFP data were analyzed using MATLAB R2018b. The recorded LFPs were filtered by a 50 Hz notching filter to remove the powerline artifact. The target oscillations  were divided according to previous studies. Raw data were transformed by Spike2 software  initially, then analyzed by using MATLAB R2018b. In order to avoid current disturbance due to DBS stimulation, the headstage of the LFP was removed from the mice as soon as the stimulation was over. The normalized power was defined as the power value, with every point divided by the mean of the baseline (before MK-801) groups.To verify the expression of adeno-associated viral functional proteins, we performed immunohistochemistry of tyrosine hydroxylase in the VTA. The mice were deeply anesthetized through the abdominal cavity with pentobarbital (100 mg/kg), and then saline was perfused through the heart. After most blood was drained out, 4% paraformaldehyde (PFA) was used for fixation. To better fix the brain tissue, the head was removed and soaked in 4% PFA at room temperature overnight. The brain was removed the next day, post-fixed overnight in 4% PFA at 4°C, and transferred to 30% sucrose in 0.1 M PBS, pH 7.4 for 24–48 h. Coronal sections (20 μm) containing the VTA were cut on a cryostat (Leica CM3050S). The slides were washed with 0.1 M PBS, pH 7.4, incubated in blocking buffer  for an hour, and then with primary antibodies  in blocking buffer overnight at 4°C. After washing three times with 0.1 M PBS, pH 7.4, the sections were incubated with donkey anti-rabbit IgG H&L  secondary antibody at room temperature for 2 h. DAPI  staining was used to identify the cell bodies. Finally, 10% glycerin was used to seal the slides. Fluorescent images were collected using a confocal microscope (Zeiss 800).p < 0.05, “ns” means no significance. Asterisks denote statistical significance . No statistical methods were used to predetermine the sample size. Unless otherwise indicated, values are reported as the mean ± SEM.The data from brain sections were processed with ImageJ software. All statistical analyses were performed with GraphPad Prism 7.0 or MATLAB R2018b. The type of statistical analysis in our study is one-way ANOVA. Data were considered statistically significant when The list of key resources is detailed in Hyperlocomotion and stereotyped behaviors, which are recognized as positive symptoms in animal models of psychosis, were chosen as the measurement indicators. Mice were first injected intraperitoneally with 0.3 mg/kg MK-801. Thirty minutes later, local electrical stimulation was started at 60 μs, 130 Hz, and 100 μA for 1 min . The behTo observe changes in the VTA before and after electrical stimulation, we first used Local Field Potential (LFP) recordings to record changes in VTA population neuronal activity. Bilateral symmetrical DBS and LFP electrodes were implanted into the VTA, and the mice were assessed after 2 weeks of recovery .Target oscillations  were classified based on previous studies. Statistical results showed significant differences in theta- and alpha-frequency waves before and after electrical stimulation of the VTA . Theta- in vivo fiber photometry to observe the Ca2+ activity of VTA neurons in real-time. Because the VTA contains 60%–65% DA neurons, 30%–35% GABA neurons, and 2%–3% glutamate (Glu) neurons, we selected DA and GABA neurons for further study.To verify the effects of DBS on VTA neurons, we used DAT-ires-cre and Vgat-ires-cre mice, respectively. MK-801 was intraperitoneally injected into mice as a pharmacological model of psychosis. The fiber recording results showed that the VTA GABA and DA neurons were rapidly activated when the electricity was turned on. Thus, these neurons showed continuous activation during electrical stimulation .


MicroRNAs (miRNAs) are regarded as a rising star in the biomedical industry. By monitoring slight increases in miRNA-21 levels, the possibilities of multi-type malignancy can be evaluated more precisely and earlier. However, the inconvenience and insensitivity of traditional methods for detecting miRNA-21 levels remains challenging. In this study, a partially complementary cDNA probe was designed to detect miRNA-21 with target-triggered dual amplification based on strand displacement amplification (SDA) and terminal deoxynucleotidyl transferase (TdT)-assisted amplification. In this system, the presence of miRNA-21 can hybridize with template DNA to initiate SDA, generating a large number of trigger molecules. With the assistance of TdT and dGTP, the released trigger DNA with 3′-OH terminal can be elongated to a superlong poly(guanine) sequence, and a notable fluorescence signal was observed in the presence of thioflavin T. By means of dual amplification strategy, the sensing platform showed a good response tomiRNA-21 with a detection limit of 1.7 pM (S/N = 3). Moreover, the specificity of this method was verified using a set of miRNA with sequence homologous to miRNA-21. In order to further explore its practical application capabilities, the expression of miRNA in different cell lines was quantitatively analyzed and compared with the qRT-PCR. The considerable results of this study suggest great potential for the application of the proposed approach in clinical diagnosis. Micro-RNA refers to a type of short non-coding endogenous RNAs with a length of approximately 22 nucleotides ,2. UbiquFrom the perspective of miRNA-21 analysis, various detective strategies and platforms have been developed . TraditiRecently, the G-quadruplex/Thioflavin T (ThT) system has been widely used for signal generation in biochemical analysis. This system can exhibit remarkable fluorescence signal enhancement ,28,29, aIn this study, to further strengthen the signal a multi-amplificative design was applied consisting of SDA and TdT-mediated amplification, with a G-quadruplex/ThT reporting system, for miRNA-21 analysis. To combine these three systems, several enzymes were used as tools. In our design, the presence of miRNA-21 can hybridize with template DNA to initiate SDA, generating a large number of trigger molecules. With the assistance of TdT and dGTP, the released trigger DNA with 3′-OH terminal was elongated to a superlong poly(guanine) sequence, so that a notable fluorescence signal was observed after ThT was added. By means of dual amplificative strategy and efficient reporting design, the sensing platform showed a good response towards miRNA-21 with a detection limit of 0.0017 nM (S/N = 3). In specificity analysis, a set of miRNAs with sequence homology to miRNA-21 was selected. In order to further explore its practical application capabilities, the expression of miRNA in different cell lines was quantitatively analyzed and compared with the qRT-PCR. All the results of this study suggest that the proposed approach has great potential in clinical diagnosis.2), were obtained from Sinopharm Chemical Reagent Co., Ltd. . DNA and RNA sequences were synthesized by Sangon Biotechnology Co., Ltd. . All other reagents were of analytical reagent grades. Ultrapure water (18.2 MΩ.cm) used in the experiments was obtained from a Milli-Q water purification system . The DNA sequences and RNA sequence list were as follows: miRNA-21, 5′-UAG CUU AUC AGA CUG AUG UUG A-3′; cDNA: ATA TCA GCG ATC ACC CAT GTT ACT CTC TAA CAG ACT CTC AAC ATC AGT CTG ATA AGC TA-3′. The reaction buffer used in this study contained two components: (1) Tris-HCl buffer , (2) 1 × TdT buffer.Terminal deoxynucleotidyl transferase (TdT), deoxy-ribonucleoside triphosphates (dNTPs), deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) were purchased from Takara Biotechnology Co., Ltd. . Klenow fragment polymerase  and the nicking endonuclease Nt.BstNBI were bought from New England Biolabs Ltd. . Tris [Tris-(hydroxy-methyl) aminomethane], hydrochloric acid (HCl), thioflavin T (ThT), sodium chloride (NaCl), magnesium chloride . The excitation wavelength was set at 425 nm and the fluorescence emission spectra were collected at wavelengths ranging from 450 to 550 nm at room temperature. All experiments were repeated at least three times. The slit widths of emission and excitation were both set at 5 nm.All experimental conditions were optimized, including the concentrations of cDNA, KF polymerase, Nt.BstNBI, TdT, and the reaction time of TdT. The concentration range of cDNA was 20–80 nM. The selected KF polymerase concentration was 50–150 U/mL. The Nt.BstNBI concentration was 20–60 U/mL. The selected concentration of TdT was 70–350 U/mL. The TdT reaction time was 120–240 min. When optimizing the experimental conditions, only one condition was changed at a time, and the other conditions remained unchanged.2, pH 7.9). The mixed buffer A was incubated at 37 °C for 40 min to form partially complementary duplex. Then, 100 U/mL KF polymerase, 40 U/mL Nt.BstNBI nicking endonuclease, and 0.1 mM dNTPs were added to the resulting solution A at 37 °C for 2 h to generate trigger DNA. Then, the digestion and the extension reaction were terminated by incubating at 80 °C for 20 min. Later, 210 U/mL TdT, 0.4 mM dATP, 0.6 mM dGTP and 20 μL 1 × TdT buffer were added and incubated in the reaction system for 180 min at 37 °C. Finally, 60 μL buffer A  and 8 mM ThT were mixed with the solution to give a final volume of 100 μL, and vigorously stirred to react at 25 °C for 10 min. The fluorescence emission spectrum was recorded at room temperature using an F-2700 fluorescence spectrophotometer.The determination of miRNA-21 was conducted as described below. Initially, 40 nM cDNA and different concentrations of miRNA-21 ranging from 0 to 200 nM were added to a mixed buffered solution containing 20 μL buffer A  from the above-mentioned different types of cells. The procedures of kit operation, sample homogenization, RNA binding (through column), column washing, and RNA elution to extract RNA were strictly followed. Then, the RNA extracted from the above cells was dissolved in DEPC water, and the extraction solution was used to detect miRNA-21 by the newly developed detection method constructed in this article and by the traditional qRT-PCR method. The protocol for performing qRT-PCR was: denaturation , and 40 amplification cycles . Each assay was performed in triplicate.In this study, a dual amplificative system for miRNA assay was developed by combining SDAs and TdT-mediated signal-on fluorescence reporting. As depicted in The design principle of this work can extensively improve the signal based on the following facts: (1) A recycling process was involved in SDA. The recycling part of target is not merely miRNA-21, the analyte, which is the single-strand RNA and may be easily degraded under improper conditions. Instead, upon the reaction, the cDNA captures miRNA-21 and its hybrids, making sure that the analyte is unlikely to be degraded during the following reactions and trigger the following amplification. (2) A dual amplificative design consisting of SDA and TdT-mediated elongation reaction was applied. The enzymes involved in this system play key roles in amplification, and their high specificity ensures that the amplification to be worked is in order and free of interference. (3) The G-quadruplex/ThT complex functions as the signal reporting system, and it has superior signal intensity compared to other label-free methods, further strengthening the signal indicating ability of this system.The feasibility of the proposed method was verified by measuring the fluorescence emission spectra of the miRNA-21 detection system under different conditions. As shown in As shown in 2 = 0.9891). The linear correlation equation is Y = 28.889X + 45.408, where Y represents the fluorescence intensity and X represents the concentration of miRNA-21. Finally, based on the calculation of the 3σ rule, the detection limit (LOD) of this strategy was obtained as 1.7 pM, which can compete with the majority of miRNA detection methods reported in recent years  were selected for quantitative analysis. nt years .As is mentioned above, practical sampling for miRNA-21 analysis can often be subject to interference from other analogues, especially other micro-RNA . As showThe presence of miRNA is almost ubiquitous within the human body . MethodsIn order to make our conclusions more convincing, miRNA-21 was further tested in different cells’ lyses. Three types of cells  were selected to detect miRNA-21 expression levels. According to the literature, the expression of miRNA-21 in these cells MCF-7, HeLa and HeK293 is in descending order, which is representative for intracellular samples ,38. The To sum up, a highly sensitive and selective sensor platform was developed for the detection of miRNA-21, based on isothermal strand displacement amplification strategy and the G-quadruplexe-thioflavin T system. Calculation based on the 3σ rule resulted in a low detection limit of 1.7 pM with a linear range of 0.002–10 nM. Finally, and importantly, this newly developed method for the detection of miRNA-21 was well validated in the selective assay, serum recovery assay, and actual cell sample assay. Therefore, we can predict that the method proposed in this study is able to make a definite contribution to the detection of miRNA-21, and play a potential part in early clinical diagnosis and targeted therapy of malignant tumors in the future.


Viruses, including influenza viruses, MERS-CoV (Middle East respiratory syndrome coronavirus), SARS-CoV (severe acute respiratory syndrome coronavirus), HAV (Hepatitis A virus), HBV (Hepatitis B virus), HCV (Hepatitis C virus), HIV (human immunodeficiency virus), EBOV (Ebola virus), ZIKV (Zika virus), and most recently SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), are responsible for many diseases that result in hundreds of thousands of deaths yearly. The ongoing outbreak of the COVID-19 disease has raised a global concern and intensified research on the detection of viruses and virus-related diseases. Novel methods for the sensitive, rapid, and on-site detection of pathogens, such as the recent SARS-CoV-2, are critical for diagnosing and treating infectious diseases before they spread and affect human health worldwide. In this sense, electrochemical impedimetric biosensors could be applied for virus detection on a large scale. This review focuses on the recent developments in electrochemical-impedimetric biosensors for the detection of viruses. In the last decade, several biosensors have been developed as an alternative method for the analysis of microorganisms, viruses, and toxins in food, as well as for various environmental and medical applications due to their ability of rapid analysis, reproducibility, stability, and accuracy ,2,3. As Viruses have excellent resistance and high transmissibility, as well they can mutate rapidly and recombine their genetic material, which increases the likelihood of a pandemic, especially in a globalized world . NumerouConventional methods, including an enzyme-linked immunosorbent assay (ELISA) and a polymerase chain reaction (PCR) are commonly applied to detect viruses such as the influenza viruses, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), human immunodeficiency virus (HIV), Ebola virus (EBOV), Zika virus (ZIKV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,10,11. TBy integrating a biological recognition component with a transducer, which transforms the activity of the biorecognition element into a measurable signal, biosensors represent valuable diagnostic tools for analyzing biological samples ,15,16,17This review focuses on the recent development of electrochemical impedance immunosensors and DNA- or RNA-based biosensors to detect currently circulating viruses.Electrochemical impedance spectroscopy (EIS) represents an emerging electrochemical technique ,22. In tThe EIS is based on the frequency dependence response of an electrochemical system to a small amplitude sinusoidal voltage signal and integrates the information about the capacitive and resistive properties of materials ,26,27. TE and the current I is the impedance Z (Equation (1)). In the equation, ω represents the angular frequency, t represents the time, i represents the imaginary number, and φ represents the phase angle between the potential or current signals 3−/4− is often used as a redox pair in the faradaic EIS, where the generation of electric current results from reduction or oxidation reactions among the electroactive species 3−/4− gets limited. In the process, the ss-DNA probe was hybridized with the target DNA to form ds-DNA. The helix formation induces ds-DNA release from the surface of the biosensor. The decrease in Rct is logarithmically related to the concentration of the HIV-1 gene in a range from 0.1 pM to 100 nM. The LOD of this sensor is determined at 23 fM.An impedimetric HIV-1 genosensor was devised by Gong et al. . This geAn alternative detection method for the HIV-1 gene using a label-free DNA impedimetric genosensor with gold nanoparticles (AuNPs)/carbonized glass fiber (GF) coal tar pitch electrodes (GTP) was designed by Yeter et al. . The devAptasensors are biosensors that use aptamers as biorecognition elements . Aptamer2) hybrid nanocomposite. The electrical condition for an AC impedance measurement was a frequency ranging from 1 Hz to 100 kHz with an amplitude of 10 mV. The LOD of this biosensor was determined at 0.4049 pg/mL, and its linear range was from 70 pg/mL to 400 pg/mL.Kim et al.  devised 6]3−/4−, the magnitude and phase of the impedance were measured at 54 points per decade. The LOD was determined at 0.25 HAU, and the linearity range was obtained from 0.125 to 16 HAU.Karash et al.  devised An electrochemical aptasensor for the detection of the HCV core antigen was developed by Ghanbari et al. . In thisRct value. According to this study, this occurrence caused the aptamers to alter conformation after binding to VACV, allowing the [Fe(CN)6]3−/4− to adhere to the electrode surface more freely.A design of an aptamer-based viability impedimetric sensor for viruses was presented by Labib et al. . In thisBachour Junior et al.  devised Rct is altered, corresponding to the amount of bound antigens 3−/4− is used as a redox probe in the process. The virus binds to the target bioreceptor (antibody) at the WE surface, and the redox reaction is hindered.In Several studies on EIS-based biosensors have been performed by designing immunosensors for virus detection .Rct). The biosensor was tested in an ideal buffered PBS solution where the LOD was 0.79 fM, and the linearity was obtained from 0.18 fM to 0.18 nM. Dunajová et al.  developeNidzworski et al.  devised Rct and HBsAg concentration was obtained in the range from 5 to 3000 ng/mL with a LOD of 2.1 ng/mL.Akkapinyo et al.  reported−4 to 5 × 10−3 IU/mL, with the LOD at 26 × 10−5 IU/mL.A label-based impedimetric biosensor was reported by Mandli et al. . An indiChowdhury et al.  devised Kaushik et al.  presenteCabral-Miranda et al.  designedCurrently, rapid and accurate diagnostic techniques are needed to prevent the further spread of COVID -19 disease worldwide. Due to this situation, many SARS-CoV-2 biosensors with different design protocols have been developed .5 PFU/mL.Mojsoska and Larsen et al.  devised Aydın et al.  develope6)]3−/4−. The frequency ranged from 10 MHz to 0.01 Hz, and an amplitude of 10 mV was applied. The LOD was 150 ng/mL, and linearity from 0.16 to 1.25 μg/mL was obtained.Zaccariotto et al.  devised An EIS-based biosensor with a recombinant ACE2-coated palladium nano-thin-film (Pd-NTF) was devised by Kiew et al.  to scree6]3−/4− to measure the Rct before and after the modified electrode came into contact with the positive or negative serum sample. This investigation maintained the perturbation amplitude at 10 mV over a frequency range from 10 kHz to 10 mHz.Lorenzen et al.  devised A genosensor for SARS-CoV-2 detection was reported by Avelino et al. . In thisA strategy for the detection of SARS-CoV-2 using interdigitated gold electrodes (AuIDE) with a spacing of 10 μm was presented by Ramanathan et al. . In thisFurthermore, in developing the impedimetric biosensor for SARS-CoV-2, an additional approach was taken using the peptides and matrix as biorecognition elements.Soto and Orozco  devised 3) on the SPE to imprint the complete SARS-CoV-2 viral particles within the polymer matrix to create viral complementary binding sites. Measurements in this study were taken at an AC potential of 5 mV, with a frequency range from 10 kHz to 0.1 Hz. The developed biosensor exhibited high selectivity against the tested SARS-CoV-2 and other corona and influenza respiratory viruses. The LOD and limit of quantification (LOQ) were 57 and 175 pg/mL, and the linear range was obtained from 7 to 320 pg/mL.SARS-CoV-2 detection using a matrix as a biorecognition element was reported by Hussein et al. . ResearcViral diseases, which result in more than one million new cases and hundreds of thousands of deaths each year, pose a severe threat to public health. In addition, viruses can change rapidly, making people vulnerable to emerging and potentially deadly viral strains, as evidenced by the COVID -19 pandemic currently affecting the entire world . The preIn this review, recent developments in impedimetric electrochemical biosensors for the detection of various viruses such as influenza viruses, hepatitis viruses, HIV, ZIKA, EBOV, and coronaviruses are examined. Researchers have applied different approaches and inventive methods to develop these biosensors. With improvements in many areas, developed biosensing devices exhibit analytical performance comparable to conventional virus detection methods. One of their main advantages is their miniaturization ability, which enables the development of portable, adaptable, and low-sample consumption biosensors .In summary, the determination of whole viruses and their components associated with a particular disease can be beneficial in differentiating and diagnosing diseases with similar clinical symptoms . In addi


Accurate and rapid molecular diagnosis of COVID-19 is a crucial step to tackle the ongoing pandemic. The primary objective of this study was to estimate the real-world performance of the novel RT-PCR STANDARD M10 SARS-CoV-2 assay in a large number of nasopharyngeal (NP) specimens eluted in universal transport medium. The secondary objective was to evaluate the compatibility of this kit in testing NP samples eluted in an inactivated transport medium  and lower respiratory tract (LRT) specimens, which are commonly collected in critical care. A total of 591 samples were analyzed. Compared with the standard extraction-based RT-PCR Allplex 2019-nCoV (time-to-result of 270 min), the sensitivities of the STANDARD M10 were 100% (95% CI: 98.1–100%), 95.5% (95% CI: 91.7–97.6%), and 99.5% (95% CI: 97.2–99.9%) for ≥1 gene, the ORF1ab gene, and the E gene, respectively, while the specificity was 100% (95% CI: 98.7–100%). The diagnostic accuracy was 100% in testing both NP samples eluted in an inactivated transport medium and LRT specimens. STANDARD M10 reliably detects SARS-CoV-2 in 60 min, may be used as a POC tool, and is suitable for testing LRT specimens in the critical care setting. Detection of SARS-CoV-2 viral RNA by means of real-time reverse transcription-polymerase chain reaction (RT-PCR) is considered the gold standard for the diagnosis of both symptomatic and asymptomatic COVID-19 cases .Standard RT-PCR assays are performed under proctored conditions and require sophisticated laboratory equipment and skilled staff. Moreover, despite some recent progress, the traditional techniques are still associated with suboptimal turnaround times ,5. Quickn = 15) and ID Now SARS-CoV-2  (n = 13) were the most frequently assessed. Compared with the standard RT-PCR assays, the pooled sensitivity and specificity of the rapid molecular tests were 95.1% (95% CI: 90.5–97.6%) and 98.8% (95% CI: 98.3–99.2%), respectively [p < 0.0001).Rapid RT-PCR assays are intended to detect SARS-CoV-2 RNA in the POC and critical care settings in a reasonably short time and have become increasingly common. Indeed, a Cochrane review  identifiSTANDARD M10 SARS-CoV-2  is an all-in-one cartridge multiplex qualitative RT-PCR assay currently approved for the molecular diagnosis of COVID-19 from nasopharyngeal (NP) or oropharyngeal swab specimens. The manufacturer’s declared sensitivity and specificity for these sample types are 100% . HoweverThe present study is reported according to the STARD (standards for reporting of diagnostic accuracy studies) statement  Supplem Table S1The study was conducted between September 2021 and March 2022 at the regional reference laboratory for COVID-19 diagnostics, located at San Martino Policlinico Hospital in the Metropolitan City of Genoa . During the study period, most detections were due to the Delta and Omicron variants of concern (VOCs) .For the main study, all samples with a conclusive result in the reference test were potentially eligible. A total of 500 (200 positive and 300 negative) NP samples swabbed by means of a sterilized cotton flock and eluted in the universal transport medium (UTM)  were tested in both reference and index tests (see below). These specimens came from both community-dwelling and hospitalized subjects. The age, sex, and sample date were readily available on the sample tube, while no clinical data could be associated with a given sample. The adopted sample size was judged sufficiently powered and determined on the basis of the Foundation for Innovative New Diagnostics (FIND)  and EuroAll samples were analyzed in fresh and within eight hours of arrival at the laboratory. These were first tested by the reference standard RT-PCR assay available (see below). Once the results of the reference tests were obtained, the leftover samples were consecutively collected and tested in the STANDARD M10 assay. Both reference and index tests were performed on the same day.For the complementary evaluations of the STANDARD M10 kit, we assessed its compatibility and performance in detecting SARS-CoV-2 in NP swabs eluted in the eNAT sample collection system . The eNAT system is a guanidine-thiocyanate-based medium used for virus inactivation and is particularly useful for the POC assays performed outside biosafety level 2 . The eNAFinally, we evaluated the compatibility and performance of the STANDARD M10 kit in detecting SARS-CoV-2 in LRT specimens, including broncoaspirate and bronchoalveolar lavage samples. These sample types came from the hospital intensive care units and were collected in sterile plastic tubes. Owing to their particular viscosity characteristics, LRT samples were first pre-treated with a liquifying dithiothreitol-based solution  at a 1:4 ratio, vortexed, and left to stand for 15 min.The STANDARD M10 assay is an all-in-one cartridge-based ready-to-use multiplex  RT-PCR kit intended for the qualitative detection of SARS-CoV-2 RNA. RT-PCR is performed on the STANDARD M10 analyzer  with up to 8 scalable modules. The assay targets the ORF1ab and E genes, and the resulting amplification curves are displayed for both gene targets. Detection of the ORF1ab gene independently from the E gene precludes a “Positive” result, while samples for which only the E gene is detected are deemed “Presumptive positive”. “Invalid” results are displayed when the internal control was not amplified or target signals did not have a Ct within the valid range . ProvidiTesting on the STANDARD M10 was performed according to the manufacturer’s instructions . BrieflyFinally, the LOD for the eNAT samples was determined by using the AccuPlex SARS-CoV-2 reference kit , which contains positive reference materials for the whole genome at a concentration of 5000 copies/mL. A total of six serial dilutions  were prepared using the eNAT liquid and the positive control. Each dilution was tested in six replicates.Reference RT-PCR assays used in the present study were those available in the laboratory for routine diagnostics. For the main study of testing NP swabs eluted in UTM, the standard extraction-based qualitative RT-PCR Allplex 2019-nCoV  was used. The whole procedure followed the instructions provided by the manufacturer .The Alinity m Resp-4-Plex kit  was used as a reference test for LRT specimens and as a resolver test for NP swabs eluted in UTM and eNAT. This assay is a multiplex RT-PCR for use with the automated Alinity m system  for the qualitative detection and differentiation of RNA from SARS-CoV-2, influenza A and B, and respiratory syncytial viruses. Concerning SARS-CoV-2, the RdRp and N genes are targeted, and positive results are displayed as a unique Ct value. According to the manufacturer, the LOD is 30 genome equivalent units/mL, and the time to the first result is <115 min .Finally, for NP swabs eluted in the eNAT system, two different all-in-one cartridge-based rapid RT-PCR assays were used, namely Vivalytic SARS-CoV-2   and Novor coefficient was computed to establish a correlation between Ct values for the E gene (being the only common target) provided by the index and reference tests. Paired t-tests were used to compare average E gene Ct values. The relative diagnostic accuracy of the index vs. reference standard tests was quantified by the overall accuracy, sensitivity, specificity, and Cohen’s κ. Hypothetical positive (PPV) and negative (NPV) predictive value curves were constructed from the observed sensitivity and specificity and by varying the disease incidence from 0 to 50%. Probit regression was used to estimate the 95% LOD for the eNAT samples.Categorical variables are expressed as proportions with 95% CIs, while continuous variables are expressed as means ± standard deviations or medians with interquartile ranges (IQRs). No imputation was performed since no missing data occurred. Pearson’s Statistical analysis was carried out using R stat packages v. 4.1.0 .A total of 200 positive and 300 negative NP swabs eluted in UTM were analyzed in both Allplex 2019-nCoV and STANDARD M10 assays . The medA total of 9 of 500 samples  produced invalid results in the first STANDARD M10 run and were repeated. The second run was successful for all nine samples. The overall relative accuracies of the STANDARD M10 assay were 99.8 and 98.2% for the E and ORF1ab genes, respectively. The corresponding sensitivity parameters were 99.5 and 95.5%, respectively. When considering the detection of at least one gene target, the between-assay agreement was perfect, with a relative sensitivity of the STANDARD M10 of 100%. No false-positive results emerged, giving a specificity of 100% .As the specificity was 100%, the PPV was 100% independent of the SARS-CoV-2 positivity prevalence. As shown in p < 0.001) than those determined by the STANDARD M10 kit (mean 21.8 ± 6.1), confirming the lower LOD of the latter. The E gene Ct values determined by both assays were highly correlated (p < 0.001), with a Pearson’s r of 0.954 (95% CI: 0.939–0.965)  were, on average, 2.2 (95% CI: 2.0–2.5) points higher (9–0.965) .For the first complementary study, a total of 30 positive and 30 negative eNAT NP samples were tested in both STANDARD M10 and reference tests. All positive patients were also positive in the standard Allplex 2019-nCoV assay performed on samples collected in the UTM system. The reference test for the first 23 samples was the Vivalytic SARS-CoV-2 assay, while the remaining 37 samples were processed using the Novodiag COVID-19 kit. The agreement between the STANDARD M10 and Vivalytic SARS-CoV-2 was perfect (100%). By contrast, concerning the comparison between the STANDARD M10 and Novodiag COVID-19, there was one discordant result. In particular, this eNAT sample was negative for both gene targets in the Novodiag COVID-19 assay but positive for both the ORF1ab (Ct 33) and E (Ct 31) targets in the STANDARD M10 kit. This sample was therefore tested in the Alinity m Resp-4-Plex assay, and the result was positive (Ct 32). The rate of invalid results was identical  for both index and reference tests; because of the limited sample volume, these eNAT samples could not be retested. The overall accuracy, sensitivity, and specificity of the STANDARD M10 assay were thus 100% (95% CI: 93.7–100%), 100% (95% CI: 87.5–100%), and 100% (95% CI: 88.7–100%), respectively. The estimated LOD to achieve a 95% detection rate for NP swabs eluted in eNAT was 235 copies/mL  Table S2Finally, a total of 31 LRT samples were tested. Of these, 9 and 22 were determined as positive and negative in the Alinity m Resp-4-Plex assay, respectively. In the first STANDARD M10 run, there was one invalid result (3.2%), which turned positive in the second run. In the STANDARD M10 assay, the ORF1ab and E genes were detected in 88.9% (8/9) and 100% (9/9) of samples, respectively. The corresponding sensitivity parameters for ≥1 target, the ORF1ab gene, and the E gene were 100% (95% CI: 70.1–100%), 88.9% (95% CI: 56.5–98.0%), and 100% (95% CI: 70.1–100%), respectively. The only “Presumptive positive” results had Alinity m Resp-4-Plex Ct values of 35. No false-negative results were observed .This study is the first in-field study to evaluate the diagnostic performance of the novel RT-PCR kit STANDARD M10 for the rapid molecular diagnosis of SARS-CoV-2 in NP specimens with a wide range of viral loads. Generating real-world evidence on the performance of diagnostic tools for SARS-CoV-2 has become increasingly important . The obsIn the main study, the sensitivity for at least 1 gene target and the specificity were both 100%. On the other hand, the estimated sensitivity of the STANDARD M10 was higher for the E gene than for the ORF1ab gene; indeed, the latter was not detected in 4.5% of positive samples. A significant proportion of the ORF1ab dropout has been reported for both Alpha  and DeltThe handling and processing of specimens present a certain level of biohazard. In this regard, immediate viral inactivation prior to sample processing is an ideal solution for the POC setting since chemical inactivation may eliminate any risk of aerosol or droplet generation ,29. In tWe then demonstrated that the STANDARD M10 assay may be also used for the efficient detection of SARS-CoV-2 in LRT specimens. Critically ill patients are often under invasive mechanical ventilation, and the collection of upper respiratory tract samples is usually not possible under these circumstances. Moreover, testing LRT samples may be useful in patients under clinical investigation whose NP swabs are negative . To our Finally, the observed “invalid” result rate was relatively low (1.8%). In our opinion, the rate of invalid results should always be recorded in validation studies as well as product-related materials. Indeed, high invalid result rates may compromise both the speed and efficiency of laboratory diagnostics  and the product cost-effectiveness profile.The main limitation of the present study is that we were not able to link the RT-PCR output to patients’ clinical characteristics, such as the presence or absence of symptoms or days since the onset of symptoms. Indeed, our evaluation was conducted under real-world conditions, and such information is often unknown to the laboratory personnel. However, we believe that this shortcoming has a limited impact on the conclusions since the STANDARD M10 index test results were highly congruent with those obtained by the routinely used standard RT-PCR assay targeting three genes. Second, the two complementary studies were designed as feasibility evaluations of the compatibility of the index test with alternative collection kits and specimen types and are thus not sufficiently powered to estimate the false-positive and false-negative rates. Larger in-field evaluations of the STANDARD M10 assay on NP swab samples eluted in the eNAT or similar inactivating systems and LRT specimens are warranted. Third, the present study included only samples positive for the two most recent Delta and Omicron VOCs, and therefore, our findings may be not generalizable to both previously circulating and future viral populations. Indeed, considering a continuous evolution of SARS-CoV-2 , the perIn conclusion, the STANDARD M10 assay is a reliable kit for the rapid molecular diagnosis of SARS-CoV-2 infection, may be used as a POC tool, and is suitable for testing LRT specimens in the critical care setting.


Aims: The LEADER trial demonstrated that the glucagon-like peptide-1 receptor agonist (GLP1-RA) liraglutide reduces kidney and cardiovascular (CV) risk in patients with type 2 diabetes. We previously developed a Parameter Response Efficacy (PRE) score that translates multiple short-term risk marker changes, from baseline to first available follow-up measurement, into a predicted long-term drug effect on clinical outcomes. The objective of this study was to assess the accuracy of the PRE score in predicting the efficacy of liraglutide in reducing the risk of kidney and CV outcomes.Methods: Short-term changes in glycated hemoglobin (HbA1c), systolic blood pressure (BP), urinary-albumin-creatinine-ratio (UACR), hemoglobin, body weight, high-density-lipoprotein (HDL) cholesterol, low-density-lipoprotein (LDL) cholesterol, and potassium were monitored in the LEADER trial. Associations between risk markers and kidney or CV outcomes were established using a multivariable Cox proportional hazards model in a separate pooled database of 6,355 patients with type 2 diabetes. The regression coefficients were then applied to the short-term risk markers in the LEADER trial to predict the effects of liraglutide on kidney (defined as a composite of doubling of serum creatinine or end-stage kidney disease) and CV  outcomes.Results: Liraglutide compared to placebo reduced HbA1c (1.4%), systolic BP (3.0 mmHg), UACR (13.2%), body weight (2.3 kg), hemoglobin (2.6 g/L), and increased HDL-cholesterol (0.01 mmol/L) . Integrating multiple risk marker changes in the PRE score resulted in a predicted relative risk reduction (RRR) of 16.2% (95% CI 13.7–18.6) on kidney outcomes which was close to the observed RRR of 15.5% (95% CI -9.0–34.6). For the CV outcome, the PRE score predicted a 7.6% (95% CI 6.8–8.3) RRR, which was less than the observed 13.2% (95% CI 3.2–22.2) RRR.Conclusion: Integrating multiple short-term risk markers using the PRE score adequately predicted the effect of liraglutide on the composite kidney outcome. However, the PRE score underestimated the effect of liraglutide for the composite CV outcome, suggesting that the risk markers included in the PRE score do not fully capture the CV benefit of liraglutide. In the last years, glucagon-like peptide 1 receptor agonists (GLP1-RA) including liraglutide, semaglutide, albiglutide, dulaglutide, and efpeglenatide have been shown to decrease the risk of cardiovascular (CV) events in patients with type 2 diabetes and atherosclerotic CV disease or at high CV risk . In addiEmerging studies show that the improvement in glycemic control only mediates a small part of the kidney protective effect of GLP1-RA. In the LEADER and the SUSTAIN-6 trial, the reduction in HbA1c with liraglutide and semaglutide only explained 25% of the kidney protective effect of these therapies . GLP1-RARecognising the multiple effects of GLP1-RA in mediating kidney and CV protection, we hypothesized that a multivariable risk score, which integrates the short-term effects of drugs, would outperform single surrogate in the prediction of drug effects on long-term clinical outcomes. The multiple Parameter Response Efficacy (PRE) score was previously developed to predict the long-term effect of angiotensin receptor blockers, endothelin receptor antagonists, and sodium glucose co-transporter 2 (SGLT2) inhibitors on kidney and CV outcomes . In the The PRE score was used to estimate the effect of liraglutide on kidney and CV outcomes. The PRE score was intended as a flexible algorithm which can be used to any drug or population by fitting the regression coefficients from the multivariable Cox proportional hazards model onto the short-term risk markers measured in an independent population. In this study, the short-term risk markers and outcome relationships were established at baseline in a background dataset consisting of a pooled population of patients with type 2 diabetes at high risk of kidney events or with an established CV disease from the ALTITUDE, RENAAL, and IDNT trials . The pooIn addition, we selected patients from the LEADER trial who fulfilled the inclusion criteria of the ongoing FLOW trial which is designed to assess the effect of semaglutide on major kidney and CV death outcomes in patients with diabetic kidney disease (NCT03819153). In this subgroup from the LEADER trial, we estimated the effect of liraglutide on the composite kidney and CV death outcomes to predict future results of the FLOW trial.Variables that were measured in the intention-to-treat population in the LEADER trial and previously identified as risk markers for kidney or CV outcomes were used, i.e., glycated hemoglobin (HbA1c), systolic blood pressure (BP), urinary-albumin-creatinine ratio (UACR), body weight, hemoglobin (Hb), high-density-lipoprotein (HDL) cholesterol, low-density-lipoprotein (LDL) cholesterol, and serum potassium (K).Since the PRE score was initially developed to predict drug efficacy on kidney outcomes, we first predicted the effect of liraglutide on the kidney outcome which was a secondary outcome in the LEADER trial and defined as a composite of confirmed doubling of serum creatinine (DSCR) or end stage kidney disease (ESKD). The CV outcome in the LEADER trial was defined as a composite of non-fatal myocardial infarction, non-fatal stroke, or CV death. For prediction of the effect of GLP1-RA in the ongoing FLOW trial, we used the primary outcome of the trial defined as a composite of a sustained decline in estimated glomerular filtration rate (eGFR) by 50%, ESKD, or CV death.o(t) o(t)) and the sum of the linear function of the estimated β coefficients and the respective risk marker measurement(s) (X). The mean difference in the predicted risk in the liraglutide arm, adjusted for the mean difference in their predicted risk at the placebo arm, represents the PRE score and reflects an estimated kidney or CV risk reduction induced by liraglutide treatment. To estimate the 95% confidence intervals (CI) on the predicted RRR, 100 sets of coefficients were generated from independent normal distributions based on the estimated regression coefficients and their standard error from the Cox proportional hazards model.A multivariable Cox proportional hazards model was used to estimate the beta-coefficients associated with kidney or CV outcomes in the background dataset. These beta-coefficients were then applied to the baseline and 6-months (or first available) risk marker measurement for patients in the LEADER trial, to estimate the risk of specific outcomes at both time points in the placebo or liraglutide arm, h(t) = hIn the LEADER trial dataset, several risk markers were not available at 6-months, therefore we used those available at subsequent visits . To assess the impact of the selection of risk markers at different time-points, we performed an additional analysis using all biomarkers at 24-months. To further assess the robustness of our findings we also performed a complete case analysis based on the risk marker changes between baseline and first follow-up measurement .For the prediction of the FLOW trial outcomes, we performed additional simulation analyses with a simulated range of liraglutide induced responses on albuminuria, HbA1c, systolic BP, and body weight since the effect of semaglutide on the various risk markers may differ from the effects of liraglutide in the LEADER trial. We simulated these markers because they are important risk markers of kidney and CV outcomes and showed the most significant reductions following liraglutide treatment. Simulations were performed by shifting the distribution of the responses in these risk markers. This was done by selecting different proportions of patients with a response in the risk marker where response was defined as a reduction more or equal than the median.p-value smaller than 0.05 was considered statistically significant. All statistical analyses were performed in R version 4.1.1 .Based on the missing at random assumption, we imputed all variables which contain missing data using Multiple Imputation by Chained-Equation  where predictive mean matching, a semi-parametric approach that replaces missing values based on multivariable regression was used . Covaria2 and 1,982 (21.2%) patients had an eGFR <60 ml/min/1.73 m2. The median UACR at baseline was 24.8 mg/g. Demographic and clinical characteristics of the patients were well-balanced between the treatment and placebo group  or placebo  and were included in the intention-to-treat population. The participants of the LEADER trial were characterized by high CV risk in general. At baseline, there were 7,598 (81.3%) participants who had an established CV disease. The mean HbA1c was 8.7%, systolic BP was 135.9 mmHg, eGFR was 79.1 ml/min/1.73 mbo group .p < 0.001). A decrease in hemoglobin (2.6 g/L) and mild elevation of HDL-cholesterol (0.01 mmol/L) were observed (p < 0.01). The direction and magnitude of the effect of liraglutide on individual risk marker change was similar in the complete case analysis and the additional analysis using biomarker changes from baseline to 24-months (Short-term risk marker changes at 6-months (or first available) measurement observed in the LEADER trial are given in 4-months .During 3.8 years of follow-up in the LEADER trial , there were 237 (2.5%) patients who developed a kidney outcome, and 1,302 (13.9%) who developed a CV outcome. The observed RRR of liraglutide was 15.5% (95% CI −9.0–34.6) for the kidney outcome, and 13.2% (95% CI 3.2–22.2%) for the CV outcome . The prevs. 15.8% (95% CI -11.6–36.5), respectively. The PRE score underestimated the RRR for the CV outcome [predicted 7.7% (95% CI 6.9–8.6) vs. observed 13.9% (95% CI 2.4–24.0)]  4–24.0)] .In the analysis using risk marker changes from baseline to 24-months , the observed RRR for the kidney outcome was 15.5% (95% CI −9.0–34.6), while the PRE score predicted a RRR of 14.7% (95%CI 11.9–17.4) . For the2 and median UACR was 1,086.9 mg/g (p < 0.001 (There were 327 (3.5%) patients from the LEADER trial who met the inclusion criteria of the ongoing FLOW trial. In this subgroup, the mean age was 66.7 years, mean eGFR was 38.9 ml/min/1.73 m6.9 mg/g . The bas < 0.001 .Using the PRE score, we estimated that semaglutide as compared to placebo in the FLOW trial will lead to a RRR of 16.3% (95% CI 5.9–25.7) for the composite kidney or CV death outcome . Our simIn this study, we demonstrated that integrating short-term changes in multiple risk markers resulted in a predicted RRR of 16.2% for the kidney outcome which was of similar magnitude to the RRR observed in the LEADER trial. These results suggest that integrating short-term effects of liraglutide on multiple cardiorenal risk markers adequately predicts its long-term efficacy on the kidney outcome. The PRE score underestimated the CV protective effect of liraglutide suggesting that mechanistic pathways beyond those captured by the risk markers included in the PRE score may explain the CV protective effect of liraglutide.GLP1-RA are originally developed as glucose-lowering agents for the treatment of type 2 diabetes. GLP1-RA are approved by regulatory agencies based on their HbA1c lowering efficacy . HoweverWhich other mechanisms could explain the long-term protective effects of GLP1-RA? The mechanisms underlying the long-term protective effects of incretin-based therapies are not completely understood but several possibilities exist. It is well known that GLP1-RA decrease multiple CV risk markers. These beneficial effects may explain long-term CV protective effects. However, a mediation analysis from the LEADER trial demonstrated that the CV protective effect of liraglutide was only partly mediated by traditional risk markers suggesting, as in the current analyses, that mechanistic pathways not captured by routinely used clinical chemistry markers may be involved . Indeed,In our study, albuminuria reduction represented a strong predictor of the long-term CV and kidney effect of liraglutide. Earlier studies with angiotensin receptor blockers, endothelin receptor antagonists, and sodium glucose co-transporter 2 inhibitors have shown that albuminuria reduction is strongly associated with subsequent reductions in major kidney outcomes. . There iThe FLOW trial is the first dedicated trial to assess the effect of a long-acting GLP1-RA for kidney or CV death protection among type 2 diabetes patients with varying degree of kidney impairment and albuminuria status . We estiThere are several limitations in our study. First, the effect of liraglutide on the kidney and CV outcomes showed a wide confidence interval which limited our ability to make direct comparisons of the PRE score predicted effect with the observed treatment effect. Additional kidney and CV outcome studies with GLP1-RA, such as the FLOW and SOUL (NCT03914326) trials with semaglutide, are therefore required to more definitively confirm the validity of the PRE score. Second, to keep the PRE score relatively easy to apply, it focused on modifiable risk factors and does not account for changes in non-modifiable risk factors , changes in medication use, or complex interactions between risk factors. Third, not all parameters were available in the 6-months and thus we have used the baseline and first available follow-up measurement to account for the drug-induced risk marker changes, with additional analyses conducted with the complete cases and risk markers measured at baseline and 24-months. Fourth, the primary endpoint for the FLOW trial is based on a relatively small subgroup as we note that the majority of the participants in the LEADER trial had only mild-to moderate chronic kidney disease. The analysis is thus exploratory and post-hoc in nature and therefore can only be considered hypothesis generating. Finally, the background population consisted of patients with different characteristics than those enrolled in the LEADER trial. However, we have previously shown that this does not impact the predictive performance of the PRE score .In conclusion, integrating multiple short-term GLP1-RA induced drug effects in an algorithm (PRE score) led to a prediction of the effect of liraglutide on major kidney outcomes which was similar as the observed drug effect in the LEADER trial. However, the PRE score underestimated the effect of liraglutide on CV outcomes. Additional research is warranted to determine if adding novel biomarkers to the PRE score would improve prediction of the CV protective effects of GLP1-RA.


Thermostable exoshells (tES) are engineered proteinaceous nanoparticles used for the rapid encapsulation of therapeutic proteins/enzymes, whereby the nanoplatform protects the payload from proteases and other denaturants. Given the significance of oral delivery as the preferred model for drug administration, we structurally improved the stability of tES through multiple inter-subunit disulfide linkages that were initially absent in the parent molecule. The disulfide-linked tES, as compared to tES, significantly stabilized the activity of encapsulated horseradish peroxidase (HRP) at acidic pH and against the primary human digestive enzymes, pepsin, and trypsin. Furthermore, the disulfide-linked tES (DS-tES) exhibited significant intestinal permeability as evaluated using Caco2 cells. In vivo bioluminescence assay showed that encapsulated Renilla luciferase (rluc) was ~3 times more stable in mice compared to the free enzyme. DS-tES collected mice feces had ~100 times more active enzyme in comparison to the control (free enzyme) after 24 h of oral administration, demonstrating strong intestinal stability. Taken together, the in vitro and in vivo results demonstrate the potential of DS-tES for intraluminal and systemic oral drug delivery applications. Goblet cells, endocrine cells, Paneth cells, and specialized M cells on the GI tract may likewise perform specialized roles in drug adsorption offering unique characteristics for oral drug development [Oral is the most preferred and convenient mode among all drug administration routes and is the dominant mode of drug administration for common medicines. Oral delivery offers the best patient compliance as drugs are self-administered with flexible dosages following the patients’ needs ,2,3. In elopment ,5. Althoelopment ,6,7.Proteins and peptides are widely used in clinical care with only <5% administered orally, mostly due to the instability at pH variations and degradative enzymes in the stomach and proximal small intestine . NanoparArchaeoglobus fulgidus ferritin. They are 12 nm in diameter with an 8 nm aqueous cavity, a volume that can theoretically accommodate protein(s) of molecular volume ~270 nm3. The shells contain four 4.5 nm surface pores, which allow small molecule substrates to permeate while blocking the interaction of macromolecules such as antibodies and proteases [We recently developed a novel protein cage ‘tES’ by engineering the thermostable roteases . The advroteases , stabiliroteases , and serroteases .Here, we describe the structural refinement of tES by incorporating multiple inter-subunit disulfide linkages to create a disulfide-linked tES with practicable advantages in oral delivery applications. We observed that by engineering natural covalent crosslinking using molecularly precise disulfide linkages, ie DS-tES, we were able to protect encapsulated enzymes against the adverse GI environment when compared to tES and/or enzyme alone. Furthermore, an in vitro Caco2 assay exhibited permeabilization through intestinal epithelial cells, confirming enhanced absorption. Finally, rluc encapsulated within DS-tES displayed clear protein stabilization throughout all sections of the GI tract via the clear presence of bioluminescence in feces following oral administration in mice. To the best of our knowledge, this is the first report of an engineered protein shell with applications in the oral delivery of protein cargos. Previously, we reported the application of tES in delivering an iron-mediated catalytic center for intratumoral bioorthogonal catalysis and regression of solid tumors . To exteAs indicated in Acidic gastric fluid, together with enzymatic barriers such as pepsin and trypsin, is biologically designed to degrade proteins and thus prohibits the administration of most protein-based therapeutics. We thus evaluated the stability of HRP as a model peptide encapsulated within the DS-tES (DS-tES-HRP) in a simulated GI environment, and the activity was compared to free HRP or tES-HRP (non-disulfide stabilized). In the presence of pepsin and pH 4.0, DS-tES-HRP exhibited ~5 times and ~14 times more enzymatic activity compared to tES-HRP or free HRP, respectively a. HoweveTo assess transcytosis of DS-tES through intestinal epithelium, a permeability assay was carried out in human colon carcinoma cell line Caco2 as it represents the best available in vitro model of small intestine enterocytes ,37. As ep < 0.001) was observed which protected rluc activity for the length of the GI tract up to 24 h. The luciferase activity was then reduced ~6 times from 24 h to 72 h. On the contrary, the free luciferase activity was reduced to more than 50 times after 24 h, suggesting that DS-tES could protect and maintain the luciferase activity upon oral administration , digestive enzymes , and mucosal and cellular barriers, that collectively affect the structure-function of the loaded therapeutic proteins or peptides. Our study demonstrates that DS-tES protect encapsulated substrates in this environment a–c. It i3 . Additio−1; ThermoFisher, Singapore) resistance LB agar  plates and subsequently sequenced for positive confirmation. The positive constructs were transformed into BL21(DE3) E. coli-competent cells. An overnight starter culture (25 mL) prepared from a single positive colony was used to inoculate LB broth (1000 mL) maintained at 37 °C and supplemented with kanamycin. The bacteria culture was grown until the absorbance (OD600) of ~0.7 was obtained. Protein expression was induced using isopropyl β-D-1-thiogalactopyranoside , and the culture was allowed to grow for another 5 h at 37 °C. The bacteria were pelleted by centrifugation at 14,000× g for 15 min. The pellet was resuspended in the lysis buffer  and incubated on ice for 15 min, followed by sonication and centrifugation. As previously described [The amino acids at the tES dimerization intersection were identified for nucleotide substitution based on interatomic distance using the PyMOL software. The PRSF plasmid carrying tES gene19 was purified using the OMEGA E.Z.N.A Plasmid DNA Mini Kit I (D6922-02). The primers for nucleotide replacement were designed using QuikChange Primer Design and the PCR reaction was performed using the QuikChange lightening Site-Directed Mutagenesis Kit 210519). The PCR product was transformed into competent cells provided by the kit. The colonies were selected using kanamycin (25 µg⋅mL0519. TheThe particle sizes of tES and DS-tES were measured by dynamic light scattering (DLS) studies . The purified tES and DS-tES were aliquoted in sized cuvettes and the measurements were achieved at a scattering angle of 90°. The formation of disulfide bonds was assessed using non-reducing SDS-PAGE. In addition, the concentration of free thiols present in the DS-tES was determined using Ellman’s Assay. Briefly, 5,5′-dithio-bis-2-(nitrobenzoic acid) or DTNB prepared at 4 mg/mL concentration in 0.1 M sodium phosphate buffer was mixed with protein samples and the generation of the yellow adduct was measured at 412 nm. A standard graph was determined using known concentrations of L-cysteine and the free thiols were quantified. Additionally, the presence of endotoxin in the purified protein samples was determined using an endotoxin kit  as per the user instructions.The DS-tES in presence of the reducing agent was acidified to pH 5.8 to obtain the subunits. The subunits were mixed with a 10-fold molar excess of HRP , and incubated at 4 °C for 30 min. The pH of the mixture was increased to 8.0 and the assembled DS-tES encapsulating HRP was separated from free HRP using a non-reducing buffer on SEC. The HRP encapsulation was analyzed using SDS-PAGE. Contrarily, DS-tES-rluc was prepared as described earlier with the above-mentioned nucleotide substitutions ,24.DS-tES-HRP/tES-HRP/HRP were incubated for 2h in the presence of 1 mg/mL pepsin. The molar ratio of HRP to pepsin used in this study was 1:100  and 3 M HCl was used to make different pH values ranging from 2 to 8 and pH was reverted to 8 by NaOH to deactivate the pepsin enzyme. The HRP activity was assayed after 2 h using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in a 96-well crystal-clear polystyrene plate (Greiner Bio-One). Absorbance was measured at 450 nm using a Perkin plate reader.DS-tES-HRP/tES-HRP/HRP were incubated for 2 h in different concentrations of trypsin (0.25 and 0.4%). The HRP activity was measured similarly to as described in the pepsin assay.2 incubator for 21 days at 37 °C. The permeability assay was performed as described before [The penetrance of the DS-tES into the epithelium of the intestine was carried out in the human colon carcinoma cell line, Caco-2. Briefly, 0.5 million Caco2 cells were seeded on transwell plates and incubated in a 10% COd before . The HRPTo investigate the stability of DS-tES and tES in vivo, 0.8 mM of DS-tES-rluc, tES-rluc, or equivalent activity of free rluc were orally administered to Balb/c mice. Followed by intraperitoneal injection of the ViviRen™ In Vivo Substrate (0.4 mM) in 3 and 24 h. The distribution of the bioluminescence was evaluated using an IVIS Spectrum. In addition, mice feces were collected to assess the presence of the enzyme in the GI tract after 5, 24, 48, 72 h, and 1 week of oral administration of DS-tES-rluc.DS-tES may be useful in oral delivery systems which encapsulate and protect protein cargos from functional destabilization. DS-tES utilize a simple and rapid encapsulation technique without affecting the properties of the encapsulate, which may have significant implications for the nutraceutical and pharmaceutical industries. Future evaluation for suitable therapeutic formulations, systemic distribution, and toxicity studies is thus justified.


EQ5D is a generic measure of health. It provides a single index value for health status that can be used in the clinical and economic evaluation of healthcare. Oxford Knee Score (OKS) is a joint-specific outcome measure tool designed to assess symptoms and function in osteoarthritis patients after joint replacement surgery. Though widely used, it has the disadvantage of lacking health index value. To fill the gap between functional and generic questionnaires with economic value, we linked generic EQ-5D-5L to the specific OKS to give a single index value for health status in KOA patients.Developing and evaluating an algorithm to estimate EuroQoL generic health utility scores (EQ-5D-5L) from the disease-specific OKS using data from patients with knee osteoarthritis (KO).This is a cross-sectional study of 571 patients with KO. We used four distinct mapping algorithms: Cumulative Probability for Ordinal Data, Penalized Ordinal Regression, CART (Classification and Regression Trees), and Ordinal random forest. We compared the resultant models’ degrees of accuracy.Mobility was best predicted by penalized regression with pre-processed predictors, usual activities by random forest, pain/discomfort by cumulative probability with pre-processed predictors, self-care by random forest with RFE (recursive feature elimination) predictors, and anxiety/depression by CART with RFE predictors. Model accuracy was lowest with anxiety/depression and highest with mobility and usual activities. Using available country value sets, the average MAE was 0.098 ± 0.022, ranging from 0.063 to 0.142; and the average MSE was 0.020 ± 0.008 ranging from 0.008 to 0.042.The current study derived accurate mapping techniques from OKS to the domains of EQ-5D-5L, allowing for the computation of QALYs in economic evaluations. A machine learning-based strategy offers a viable mapping alternative that merits further exploration.The online version contains supplementary material available at 10.1186/s13018-023-03522-0. Osteoarthritis (OA) is a chronic disease that affects the knee joint. The lifetime risk of knee osteoarthritis (KOA) is approximately 46%. Globally, 85% of the burden of osteoarthritis is attributable to KOA , making Osteoarthritis impacts every part of daily life. Associated deformity results in a rigid, unstable, and painful gait that reduces the independent walking distance and is accompanied by weight gain, sleep problems, and depression .KOA induces substantial costs. With the ageing of the population and the increasing obesity in many countries, the economic burden on healthcare systems could be even higher in the coming years . This reIn economic evaluations, generic questionnaires are preferable to disease-specific questionnaires in order to compare the value of interventions across various disease areas and programs. Most of the official national pharmacoeconomic evaluation guidelines mention EQ-5D by name as a preferred instrument for the determination of health utilities or as an illustration of a suitable instrument. Of those that did not specify a specific measure, the majority of guidelines favored calculating utilities using national preference weights, which are generally derived from societal preferences for health states .However, the Oxford knee score (OKS) was used in the majority of published studies evaluating the interventions used to treat KOA; this score has the drawbacks of being disease-specific and lacking a preference-based index value .Mapping can offer a solution when EQ-5D scores are unavailable for interventions of interest, where health-related utilities can be generated from another measure of health outcomes [Two strategies are used in mapping studies: direct utility mapping and indirect response mapping. The EQ-5D index value (utility) is predicted using direct mapping, whereas the responses to EQ-five domains are predicted using response mapping. Although response mapping requires an additional step to estimate the expected index value using available EQ-5D tariffs, indirect mapping allows for the prediction of EQ-5D-5L utility values for any country. Direct mapping would only be applicable for the country that produces the tariff .This study aims to develop indirect mapping algorithms that can predict responses to the five domains of EQ-5D based on OKS values. Utility values can be then derived from the predicted responses as a separate second step using available EQ-5D tariffs.Adults over 18 years old with KOA based on clinical and radiographic characteristics, with or without total knee arthroplasty (TKA), were included in the samples. Patients whose conditions prevented them from completing the questionnaires were excluded . The Institutional Review Board of Medical Research Institute has granted ethical approval following U.S. Department of Health and Human Services (IORG 0008812) guidelines and other applicable regulations. The research adhered to the Declaration of Helsinki's principles.n = 115) was collected to assess the generalizability of the developed model. By recruiting patients at different times, we aimed to have structurally different samples [Two cross-sectional samples were collected: estimation and external validation samples. For the estimation sample, 456 (80% of the whole sample) were recruited between December 2020 and May 2021 and used to develop the model. From September to October 2021, the external validation sample . Each response level ranges between 0 and 4. The ratings ranged from 0 to 48, with 48 representing the best health . In Egypt, the questionnaire was translated and validated. (9) The score was classified as very mild (40 to 48), mild (30 to 39), moderate (20 to 29), and severe (0–19)  .The EQ-5D-5L questionnaire evaluates health status in five domains: mobility, self-care, routine activities, pain/discomfort, and anxiety/depression. Each response level ranges between 1 and 5. In addition, the patient is required to record their overall health status using a visual analogue scale (EQ-VAS). Following assessment, the scores from the descriptive component can be reported as a five-digit number, known as profile scores. There are a total of 3,125 profile scores for EQ-5D-5L, ranging from 11,111  to 55,555 . The profile score can be converted into a utility index using a country-specific value set. Two types of value sets are available for many countries: valuation value sets, which were generated using a time trade-off (cTTO) valuation technique supplemented by a discrete choice experiment (DCE), and cross-walk value sets, which were generated by mapping between the EQ-5D-5L and EQ-5D-3L descriptive systems . All couUsing a self-administered questionnaire, the following data was gathered:Spearman's rank correlation was used to determine the conceptual overlap between the domains of EQ-5D-5L and the 12 questions of OKS. The similarity between the two measurements was examined using an exploratory Ordinary Least Squares (OLS) model, where the dependent variable was EQ-5D-5L, and the regressor was the total OKS score.Method of model selection, building, and evaluation Fig. .Fig. 1SuFour classes of ordinal models were evaluated: two regression models  and two tree-based models  and ordinal forests (OF)). The binomial and multinomial models were ruled out because they disregard information about the outcome's order. Each model class can accept distinct structures with varying performance. The structure of a model is determined by its hyperparameters and their values. Box Which predictors are incorporated into the model, and in what form have a substantial impact on its predictive performance. Consequently, each model structure was constructed using four distinct sets of predictors  (1) all predictors; 2) REF-based significant predictors; (3) model-based significant predictors; and (4) principal components. Box  REF-baseWe employed 5 × threefold cross-validations to determine the optimal model structure based on model trials. The estimation sample was divided into five non-overlapping folds, with each fold serving as an internal validation set to assess the accuracy of the model developed using the other four folds. This was repeated three times for every model structure and predictor set. Each model's cross-validated accuracy rate was the mean of the 5 × threefold accuracy rates. The optimal model structure was the one with the highest accuracy rate when cross-validated.After identifying the optimum model structure and best set of predictors in the previous step, the whole estimation sample was used to estimate the model parameters. Model parameters specify how to calculate the outcome from the predictors. They are estimated by optimizing the model's fitness for the estimation sample Table .Table 1SComparing the accuracy of no model (baseline accuracy) to the accuracy of the best model.The base level of precision for each domain is the proportion of the most prevalent level. . The cruEstimating the performance of models in terms of errors in measuring predicted utility valuesn=39) and eq5d R package [The levels in the five domains were combined to determine both the actual and predicted profile scores. Actual and predicted utilities were estimated using available tariffs  statement to improve the clarity, transparency, and thoroughness of mapping study reporting .Model building and evaluation were conducted using caret R package .The estimation sample had a mean age of 47.6 ± 13.3 years, and 321 (70.4%) were female. OA lasted an average of 6.7 ± 6.5 years. About 26% of patients complained of back pain, with hypertension being the most common comorbidity  were female. On average, osteoarthritis lasted 6.7 ± 6.5 years. About 28% of patients complained of back pain, with hypertension being the most common comorbidity , self-care (57%), usual activities (30%) and anxiety/depression (38.3%), and level 3 for pain/discomfort (33%). The average EQ-VAS was 69.3 ± 21.3.They expressed 67 different EQ-5D-5L health conditions, with utilities ranging from − 0.732 to 1  contributed to mobility. Eleven, eight, and seven questions pertained to usual activities, pain/discomfort, and self-care, respectively. Only three questions contributed to anxiety/depression.RFE ranks predictors based on their contribution to every domain , and errors in predicted utility values were calculated. The average MAE was 0.098 ± 0.022, ranging from 0.063 to 0.142, and the average MSE was 0.020 ± 0.008 ranging from 0.008 to 0.042 . Tuning models' hyperparameters permitted to proceed with 133 different model structures. The use of four different sets of predictors per every model structure increased the number of models tried to 532. Machine learning in mapping was introduced by one study which used a deep neural network (DNN) in mapping from MacNew Heart Disease Health-related Quality of Life questionnaire (MacNew) onto country-specific EQ-5D-5L utility scores. While tAnother strength is that we assessed the uncertainty around the estimated MAE using bootstrapping which does not depend on assumptions.Although machine learning algorithms result in accurate predictions using small sample size, they act as black boxes where the process of prediction is not as clear as regression analysis with known coefficients.Furthermore, mapping is not a substitute for including the EQ-5D in future studies and does not overcome the limitations of either instrument .The current study derived the mapping algorithm from OKS onto the five domains of EQ-5D-5L. With available EQ-5D-5L utility values, utility scores can be calculated, and the latter enables the estimation of QALYs in an economic evaluation. A machine learning approach presents a promising alternative in the mapping literature that warrants further exploration.Additional file 1. Appendix 1: EQ-5D-5L in estimation sample. Appendix 2: EQ-5D-5L in external validation sample. Appendix 3: Correlation among between domains of the EQ-5D-5L and questions of OKS. Appendix 4: Distribution of EQ-5D-5 L utility. Appendix 5: Characteristics of the Estimation sample. Appendix 6: Characteristics of the External validation sample. Appendix 7: Description of EQ-5D-5L Utility and Total OKS Score. Appendix 8: Mobility RFE important predictors. Appendix 9: Choosing of best model of Mobility. Appendix 10: Self-Care RFE important predictors. Appendix 11: Choosing of best model of Self-Care. Appendix 12: Usual Activities RFE important predictors. Appendix 13: Choosing of best model of Usual Activities. Appendix 14: Pain/Discomfort RFE important predictors. Appendix 15: Choosing of best model of Pain/Discomfort. Appendix 16: Anxiety/Depression RFE important predictors. Appendix 17: Choosing of best model of Anxiety/Depression. Appendix 18: Rankings of OKS questions across the five domains of EQ-5D-5L by recursive feature eliminations in the Estimation sample. Appendix 19: Model evaluation in whole external validation sample.